Multiple pathological conditions including: muscle trauma, surgical damage, and myopathies can lead to loss of skeletal muscle. Volumetric muscle loss (VML) is the loss of muscle resulting in unrecoverable muscle function. To date, the treatments for VML including, the muscle flap or graft, are not effective and are hindered by limited tissue availability and donor site morbidity. The creation of engineered musculoskeletal tissue with functional myotendinous junctions, tendon-bone anchor and innervation will not only restore the function of complex tissue such as muscle following traumatic injury or disease, but can also be used as a model for studying developmental biology and pharmacology. To address this need, our laboratory has developed a reproducible method for development of scaffold-less engineered skeletal muscle constructs. The engineered muscle we fabricate consists of a 3D construct with a fibroblast-produced extracellular matrix and myotubes aligned along the axis of strain. This in vitro construct can generate force either spontaneously or in response to electrica field stimulation. Engineered skeletal muscle is limited in vitro in that it does not develop beyon a neonatal phenotype in terms of strength characteristics and tissue organization. Recently, we have shown that following as little as one week of implantation, these muscle constructs develop a capillary system, an epimysium-like outer layer of connective tissue, and an increase in muscle fiber content. Upon explantation, the constructs increased maximum isometric force 245% and begins to develop the necessary interfaces needed to advance the phenotype toward adult muscle. The long term goal of our laboratory is to engineer a tissue-engineered functional skeletal muscle unit (SMU) which has functional interfaces and when implanted allows for the full recovery of native muscle forces. While our technology shows lots of promise for repair and replacement of damaged muscle, a lot of work remains to determine how to utilize this technology to obtain optimal recovery of muscle function. An additional concern regarding the use of this novel and innovative technology in humans, however, is the safety of use of stem/precursor cell-derived tissue in patients. It is imperative to demonstrate that the primary or stromal cells used for the generation of these constructs pose no long-term threat after transplantation. The main concern in the field is the ability to ascertain that no undifferentiated cells persist in the transplanted construct that might later lead to aberrant cellular behavior suc as cancer. This is a milestone driven project. The deliverable of this project will be a tissue-engineered functional skeletal muscle unit (SMU) of appropriate size and function for clinical use in situations of small muscle injuries, such as those found in the hand and face. In addition, at the end of the granting period, we will have a better understanding of the mechanism of graft incorporation and safety of the graft for translational applications. The SMU will enable the treatment of patients who have suffered traumatic skeletal muscle loss due to traumatic injuries or disease, including the repair/replacement of face and composite facial features.